Collagens are the major structural proteins in the extracellular matrix of animals and are defined by a characteristic triple-helix structural motif that requires a (Gly-Xaa-Yaa)n repeating sequence. The amino acids found in the Xaa and Yaa positions are frequently proline, where Pro in the Yaa position is post-translationally modified to hydroxyproline (Hyp) which enhances triple-helical stability. In humans, a family of at least 28 collagen types is present, each with type-specific biological and structural functions. The triple helical motif is also present in other proteins, such as macrophage scavenger receptors, collectins and C1q.
The most abundant collagens are the interstitial, fibril-forming collagens, particularly type I collagen. These collagens form the major tissue structures in animals through forming fibre bundle networks that are stabilized by specific cross-links to give stability and strength to the tissues. In contrast to the ‘major’ fibril forming collagens (types I, II and III) the ‘minor’ collagens are generally less broadly distributed and are typically found in particular tissue locations where the minor collagen may be a significant and critical component; e.g., type X collagen in hypertrophic cartilage or the type IV collagen in basement membranes.
The majority of commercial quantities of collagen have been derived from animals, such as bovine sources, but with the concern of transmissible diseases, especially bovine spongiform encephalopathy (‘mad cow disease’). Moreover, animal-derived collagen is limited in that extracted collagens cannot be designed and modified to enhance or change specific biological or functional properties. Collagens are subject to extensive post-translational modifications both prior to and after deposition in the extracellular matrix. In particular, the fibrillar collagens are subjected to intra- and inter-molecular cross-linking that continues over the life of the molecule in the extracellular space. Thus, the amount of cross-linking present in collagens is influenced by, among other things, the age and physiology of the tissue from which the collagen is harvested. These differences influence both the extractability of collagens from tissue and the biophysical characteristics of these collagens. As a result, collagens isolated from tissues exhibit significant lot-to-lot variability and, as bulk materials, are often analytically intractable.
Accordingly, attention has shifted away from isolation of animal collagen towards production of recombinant collagens. Further, the use of recombinant DNA technology is desirable in that it allows for the potential production of synthetic collagens, collagen-like molecules and triple helical proteins which may include, for example, exogenous biologically active domains (i.e. to provide additional protein function) and other useful characteristics (e.g. improved biocompatibility and stability).
Host systems such as yeast have been explored to recombinantly produce human coded collagen. However, for production of human mimics, yeast systems are complicated by the need to introduce genes for proline-4 hydroxylase to form the Hyp residues needed for stability of mammalian collagens. Typically, recombinant mammalian coded collagens are expressed in Pichia, which requires oxygen addition to get maximum hydroxylation, as well as methanol addition for induction, adding complexity to the system.
Other collagen-like material which does not require post translational modification has been sought as replacement to hydroxylated human collagen. Recently, research on bacterial genomes has indicated there are many putative bacterial proteins that contain Gly as every third residue and a high proline content, suggesting that collagen-like, triple-helical structures/domains may be present in certain bacterial derived proteins (Peng Y et al (2010) Biomaterials 31(10):2755-2761; Yoshizumi A et al (2009) Protein Sci 18:1241-1251). Furthermore, several of these proteins have been shown to form triple-helices that are stable around 35-38° C., despite the absence of Hyp. The triple helical composition has been confirmed in a number of cases. Examples include cell surface proteins on certain bacterial cells and filaments on Bacillus anthracis spores. It has been postulated that expression of such collagen-like constructs in prophages present in pathogenic E coli strains appear to be responsible for dissemination of virulence-related genes through infection (Bella J et al (2012) 7(6) PLoS 1 e37872). Furthermore, the knowledge on how amino acid sequence contributes to the structure and stability of a triple-helix motif also allows for the design of novel triple-helical collagen-like molecules that will be stable without the need for post-translational modifications.
Collagen has been used in many applications, including as a biomedical material where it has been shown to be safe and effective in a variety of medical products in various clinical applications (Ramshaw et al, J Materials Science, Materials in Medicine, (2009), 20(1) pg S3-S8). The bacterial collagen-like proteins also have appropriate characteristics for biomedical applications, including a lack of immunogenicity and no cytotoxicity.
Non-animal sources of collagen or synthetic triple helical proteins when recombinantly produced may lack the desired interactions that stabilise protein aggregates and which are normally present in native animal collagens. In part, this is because such proteins lack hydroxyproline which is a stabilising feature found within most animal collagens, and they also lack the specialised mammalian crosslinking sites. For certain applications, bacterially derived collagen-like proteins may not have the desired functionality for proposed medical or non-medical use applications, for example as a biomedical product. By way of illustration, the Scl2 collagen-like gene from S. pyogenes behaves like a “blank slate” as it shows few, if any interactions with mammalian cells. It would be advantageous if such molecules could be modified to enable their functionality to also be modified.
The ability to produce collagen-like or triple helical proteins from non-animal sources which exhibit enhanced stability would be highly desirable for all applications in which animal-derived collagen would normally be used. Furthermore, modifications which improve the stability of a recombinant collagen-like or triple-helical protein could also be exploited to further introduce a required functionality.
Accordingly, there is a need for a method that allows for specific, controlled modification of recombinantly or synthetically produced non-mammalian collagen-like or triple-helical proteins, wherein such a method allows the introduction of various functional modifications to the protein.